Project Summary/Abstract Despite their adverse environment, the question of how photoreceptor cells (PRC) and retinal pigment epithelial cells (RPEC) remain functionally unchanged for decades in healthy eyes remains to be answered. This grant program is focused on the molecular cascade by which bioactive derivatives of the essential fatty acid family member docosahexaenoic acid (DHA) induce effectors through GPC receptors to sustain protection of PRC and RPEC function. We have shown that the synthesis of one DHA-derived mediator, Neuroprotectin D1 (NPD1), is activated at the onset of homeostatic disruptions, and we have recently discovered a second set of mediators, elovanoids (ELVs; derived from precursors made by ELOVL4), that are critical in the cascade. Our next goal is to answer two important questions: Question 1. What mechanisms get DHA to the right places at the right times in order to give rise to the lipid mediators? Hypothesis 1 ? Several retinal cells [RPEC, PRC, and Mu?ller/glia (MG)] facilitate DHA uptake, retention, and metabolism via multiple pathways to support the synthesis of the homeostatic lipid mediators sustaining PRC integrity. Question 2. How does the lipid-mediator signaling sustain cellular integrity? Hypothesis 2 ? NPD1 and ELV are synthetized in the RPEC to sustain function of these cells in a hostile environment. These mediators are synthesized on demand when homeostatic disruption occurs which, in turn, activates counteracting signaling against oxidative-cell damage. Mechanisms include GPC receptor-mediated gene clusters, which regulate the expression of specific proteins that promote homeostasis and maintenance of cell integrity. Aim 1 tests the prediction that specific mechanisms and pathways facilitate the uptake and metabolism of DHA in RPECs, MGs and PRCs to sustain PRC renewal. Aim 2 tests the prediction that NPD1 and ELV elicit their bioactivity by targeting specific G-protein-coupled receptors. Aim 3 tests the prediction that NPD1 and ELV contribute to RPEC and PRC homeostasis by targeting gene clusters. Genes will be identified using inducible lentivirus constructs, AdipoR1 global KO, cell-type specific AdipoR1 conditional KO and MFRP (rd6) animal models and single-cell genomics techniques. Additional methods include LC?MS/MS- based lipidomic analysis, MALDI molecular imaging and human primary RPECs. The outcomes of this fundamental investigation will provide new information and understanding about DHA acquisition and retention, as well as of the basic molecular and genetic signals and mechanisms required to sustain retinal function. The results of this study may also define the parameters for future studies on the pathophysiology and targets for treatment of age-related macular degeneration as well as of inherited retinal degenerations.